Affiliations
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,
Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America

Affiliations
Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, United States of America

Figures

Abstract

Wound healing capability is inextricably linked with diverse aspects of physical fitness ranging from recovery after minor injuries and surgery to diabetes and some types of cancer. Impact of the microbiome upon the mammalian wound healing process is poorly understood. We discover that supplementing the gut microbiome with lactic acid microbes in drinking water accelerates the wound-healing process to occur in half the time required for matched control animals. Further, we find that Lactobacillus reuteri enhances wound-healing properties through up-regulation of the neuropeptide hormone oxytocin, a factor integral in social bonding and reproduction, by a vagus nerve-mediated pathway. Bacteria-triggered oxytocin serves to activate host CD4+Foxp3+CD25+ immune T regulatory cells conveying transplantable wound healing capacity to naive Rag2-deficient animals. This study determined oxytocin to be a novel component of a multi-directional gut microbe-brain-immune axis, with wound-healing capability as a previously unrecognized output of this axis. We also provide experimental evidence to support long-standing medical traditions associating diet, social practices, and the immune system with efficient recovery after injury, sustained good health, and longevity.

Funding: This work was supported by National Institutes of Health grants P30-ES002109 (pilot project award to SEE and EJA), U01 CA164337 (to SEE and EJA), and RO1CA108854 (to SEE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The great importance of gut microbiota in mammalian host health is only recently being recognized in full [1]–[4]. There is now substantial evidence linking naturally-occurring or experimentally-induced disturbances of the intestinal microbial communities with immune-associated and metabolic diseases, and with neoplasia [2]–[3], [5]–[7]. Accordingly, modifying the gut microbiota using dietary probiotics is now accepted as an emerging therapeutic and overall health promoting treatment [1], [3]–[4], [8]. Nonetheless, the mechanisms by which gut microbiota impart effects that expand beyond the gastrointestinal tract and become systemic are largely elusive. The most compelling data explaining such phenomena come from studies demonstrating that gut bacterial organisms and their metabolites interact with interrelated immunological [9]–[12], metabolic [2]–[4], [13] and neuroendocrine pathways [13]–[15].

During a recent study we observed that female mice fed with the lactic acid bacterium Lactobacillus reuteri (L. reuteri) show more frequent grooming activity compared to their control counterparts [16]. This aspect of maternal behavior is regulated at large by the neurohypophyseal hormone oxytocin, best known for its role in parturition and lactation. Our understanding of oxytocin’s classical role, however, has greatly expanded over the last decades to include substantial central nervous system effects on behavior [17]–[19]. Interestingly, its most recently discovered but not as well-characterized roles include interactions with body energy balance [20]–[21] and the immune system [22]–[24].

The hypothesis that the ingestion of a lactic acid bacterium could up-regulate the expression of the neuropeptide hormone oxytocin in the hypothalamus, although without precedent, has substantive indirect support. Indeed, considerable evidence suggests the presence of a “microbiome-gut-brain axis” [14]–[15], [25]–[26]. According to this reasoning, bacteria, including ‘probiotic’ organisms, initiate immune-related and neural signals that are transmitted from the gut to the CNS, either through blood circulation or directly via the vagus nerve [15], [25]–[28][29]. This crosstalk between the gut microbiota and vital regulatory components of the CNS, such as the hypothalamus and pituitary gland is thought to impact mammalian homeostasis, including both physical and mental health [2]–[4], [13], [15].

In the present study, we find that administering purified L. reuteri organisms in drinking water induces a significant up-regulation of the neuropeptide hormone oxytocin in mice. In order to probe downstream effects of this microbial mode of oxytocin up-regulation within a tractable mammalian model system, we used a well-characterized experimental model of cutaneous wound healing in mice [30]. We selected this approach primarily because our earlier cancer studies revealed unusually robust integumentary health in aged animals when they consumed purified L. reuteri[16]. In these studies, the mouse models consuming L. reuteri exhibited mucocutaneous hyperacidity, follicular anagenesis, and sebocytogenesis mimicking features of superb physical fitness in a gender-dependent manner.

Additionally, wound healing is a fundamental process with mechanisms inextricably linked with health and disease, such as hemostasis, fibrosis, tissue repair and remodeling, acute and chronic inflammation, and neoplastic tumor evolution [31]–[33]. Our findings suggest that L. reuteri impacts the host animal inflammatory response in an oxytocin-mediated fashion. The oxytocin-potent acceleration of wound healing benefit depends upon vagus nerve integrity and is transplantable using either CD4+Foxp3+ or CD4+CD45RBloCD25+ T regulatory immune cells (Tregs) to completely recapitulate the L. reuteri-induced phenomenon in Rag2-deficient recipient host animals. These findings reveal unexpected roles of oxytocin as a novel component of the gut microbiome-associated biological networks linking mental, social, and physical health, with widespread potential benefits for high quality and healthful life.

Results

Wound healing capability is the hallmark of sustained good health [31]. During our carcinogenesis studies, we discovered dramatically improved skin and mucosal health in animal subjects consuming probiotic-containing yogurt [16]. We hypothesized that microbes in the yogurt led to this improved epithelial phenotype. We tested our hypothesis using animal models consuming a purified preparation of lactic acid bacteria Lactobacillus reuteri ATCC-PTA-6475 [16], [34] suspended in their regular drinking water. To specifically assay wound healing capability, we first employed a traditional skin biopsy assay in aged six-month-old C57BL/6 wild type (wt) mice to quantify the extent to which consuming these organisms may impact wound healing. This assay applies a standardized 2.0 millimeter full thickness excision of dorsal skin [35]. Using this approach and then microscopically examining the excision site of these mice at three, six and twelve days after biopsy (Figures 1a and 1b), we found that aged mice of either gender consuming L. reuteri exhibited proper injury repair in half the time needed for control mice drinking regular water (Figs. 1c and 1d, and Fig. 2a). By measure of wound area, female mice consuming L. reuteri experienced a more marked increase in healing compared to their controls over male counterparts (Fig. 1c), but both sexes demonstrated similar improvements. This rapid wound-healing attribute of L. reuteri was not a generic feature of consuming any bacterium, as similar dosage levels of another microbe Escherichia coli strain K12 in drinking water did not significantly impact the wound healing process. (Wound closure at day 6: E.coli-treated (n = 5) vs Control (n = 5), p = 0.5476; E.coli-treated vs L. reuteri-treated (n = 5), p = 0.0079).

During these studies, we observed that the rapidly healing C57BL/6 mice receiving the L. reuteri supplementation also displayed increased grooming behaviors, matching our earlier published observations [16]. The neuropeptide hormone oxytocin has been implicated in increased mother-infant grooming [46], and has also been linked with immune health [24] and improved wound healing [47]. This led us to postulate a unifying role for oxytocin linking lactation with microbes such as L. reuteri in more efficient injury repair culminating in improved host fitness.

Finding that oral L. reuteri therapy increased circulating levels of oxytocin in our animal model, we theorized that microbes associated with milk consumption may stimulate vagal pathways as for pleasure reward during infant-maternal bonding [29]. Indeed, it was recently shown that favorable mood after consuming other Lactobacillus sps was abolished after surgical transection of vagal nerve connections to viscera [26]. To test this possibility we performed vagotomies, and found that C57BL/6 wt mice undergoing vagotomy lost the L. reuteri–induced oxytocin surge phenomenon (Fig. S4a). At the same time, skin wound closure was delayed after vagotomy surgery in wt mice consuming L. reuteri, when compared with L. reuteri-treated animals undergoing sham surgery (Fig. S4b and S4c). Knowing that oxytocin has been implicated in wound healing [47] and immune health [24], we went on to test roles for oxytocin during L. reuteri-induced wound repair events in our mouse models.

Oxytocin is required for the L. reuteri-induced boost in injury repair

Wild type mice fed L. reuteri in their drinking water exhibit enhanced wound healing over untreated controls. This effect is entirely transferable with Foxp3+ Tregs primed in a mouse host feeding on L. reuteri. In the absence of oxytocin, feeding mice with L. reuteri provides no wound healing benefit, and the CD4+CD25+CD45Rblo Tregs from these animals are unable to convey wound-healing advantages.

Discussion

We discovered that animals consuming purified lactic acid bacteria from human milk healed their wounds more than twice as fast as control animals. The associations of symbiotic gut organisms with various physiological and disease processes has been the focus of intense research over recent decades [1]–[4]. The present study, however, is the first to show that transient introduction of microbes into the gut microbiota, in this case with L. reuteri, bestows a distant extra-intestinal tissue, the integument, with accelerated wound healing capacity.

Successful healing of skin wounds requires a tightly orchestrated series of events with complex cell signaling cascades coordinating several fundamental biological processes [31]–[32]. The time course of these events was previously well-characterized in C57BL/6 mice, which have been extensively used in these types of investigations [48], [55]. With this strain of mice, we probed critical events of the stepwise experimental wound healing process in situ at 3, 6 and 12 days post-wounding. The processes of re-epithelialization and granulation tissue formation with collagen deposition and successive influx of different subsets of immune cells match the classical wound healing timeline previously described in C57BL/6 mice [33], [48], [56]. The L. reuteri-treated mice did not diverge from the histomorphological features of this paradigm. However, microscopically-recognized classical wound healing steps occurred much earlier in L. reuteri-treated mice when compared to controls. The L. reuteri-induced compression of wound healing events led to complete re-epithelialization at day 6, which coincided with increased granulation tissue maturity typified by quiescent fibroblasts, increased collagen deposition, absence of neutrophils, and increased lymphocytes including Foxp3+ regulatory T-cells. Superficially, this left the appearance of minimal scar formation in the excision wound site in animals treated with L. reuteri in their drinking water.

The increase in Foxp3+ Tregs in L. reuteri-fed mice matches previous reports showing that lactic acid bacteria stimulate the development of regulatory-T cells in vitro as well as in vivo[57]–[59]. It is reasonable that Foxp3+ Tregs contribute to the rapid clearance of neutrophils in the wound environment [60]–[61]. Although neutrophils are essential immediately after injury, their further persistence delays wound healing [62]–[63]. Our experiments demonstrate that depletion of CD25+ cells abolishes the beneficial effect of L. reuteri in wound healing. We also show that the depletion of the pro-inflammatory cytokine IL-17A, a well-known target of Treg anti-inflammatory activity [44]–[45], contributes to more rapid wound closure [64]. Finally, adoptive transfer of CD4+CD25+ Tregs is sufficient for the wound healing benefit. These results support a causal role for regulatory T-cells in integumentary wound healing. Further studies are required to clarify whether specific priming of Treg cells with certain bacteria is required for this beneficial effect and how the molecular profile of these cells changes under different exposure conditions.

An interesting corollary to accelerated healing properties exhibited in the present study is that feeding L. reuteri in mice also induces an anagenic shift in their hair follicles [16]. A recent study suggests that skin wounds made during the anagen stage show accelerated re-epithelialization [64]. Together, these studies reveal that reduction in inflammatory cell infiltration is the most probable explanation for rapid wound closure [64]–[65]. Thus, the L. reuteri anagen-inducing and anti-inflammatory properties may overlap as explanations of the accelerated wound healing phenomenon.

We observed an up-regulation of the neuropeptide hormone oxytocin following administration of purified L. reuteri organisms in drinking water. This finding, if translatable to humans, may be particularly important given the expanding roles of oxytocin in CNS-related functions, particularly social memory and attachment, sexual and maternal behavior, aggression, human bonding and trust, learning ability, anxiety, feeding behavior and pain cognition [17]–[19], [46], [66]–[68]. During early life, oxytocin regulates neurotransmitter gamma-aminobutyric acid (GABA) signaling in the central nervous system [69] providing a favorable mood reward [26] for social and gluttary gratification. Emerging data connect oxytocin with metabolism [20]–[21] and the immune system [22]–[24], providing satisfying evidence for its role as a global regulatory hormone. This finding not only connects microbial symbionts with social and immune fitness in evolutionary success, but also highlights enormous medical utility of microbe-induced oxytocin.

Because wound healing ability serves as a barometer for systemic health and fitness [31], we tested the role of oxytocin by studying wound healing capabilities of knockout mice at 6 days after wound induction. Delayed wound healing in oxytocin-deficient animals accompanied significant increases in neutrophils, mast cells, and Il-17+ macrophages, with decreased Foxp3+ cells, indicating an abnormally persistent reaction. Both wild type and oxytocin-deficient animals carried occasional oxytocin-receptor positive immune cells. Likewise, oxytocin-deficient mice had Foxp3+ cells within their convalescing wounds, indicating that Treg cells are present but unable to offer L. reuteri-induced wound repair assistance in the absence of oxytocin.

Oxytocin-mediated lymphocyte activation due to oral L. reuteri treatment could be an indirect or perhaps direct effect, since lymphocytes have been reported to possess a cell membrane receptor for oxytocin [17], [22]. In our studies using immune-competent mice, cells of skin innate immunity, such as neutrophils and mast cells, were reduced within wounds at the 6-day time-point as a result of the L. reuteri-induced, Treg-associated, oxytocin-mediated effect. This result matches a previous study in which the increased capacity of oral tolerance in mice to heal their skin wounds correlated with decreased numbers of mast cells and neutrophils in their wounds. That study concluded that excessively robust neutrophilic and mast cell immune responses may have slowed-down the healing process [51].

Our results suggest the presence of a multi-directional microbiome-gut-brain-immune system network in which oxytocin plays a pivotal role [14]–[15], [25]–[26], [70]–[71]. The vagus nerve serves as a bidirectional communication channel between the gut and the brain [26]–[29], and is required for gut-brain signaling initiated by L rhamnosus upregulating central GABA receptor expression [26]. The full L. reuteri-induced up-regulation of oxytocin and rapid wound repair required an intact vagus nerve as suggested by the unimproved wound healing phenotypes in vagotomized mice.

Our previous work and that of many others revealed that CD4+ cells are a basic cellular carrier of microbially-triggered signals that shape the immune system to restore homeostasis in inflammatory-associated pathologies, cancer, and reproductive health [6][9], [42], [45], [70], [72]–[75]. We discovered that the L. reuteri benefit was transferable using CD4+ cell subsets alone, as long as those cells were from oxytocin-potent donors. In our previous studies we showed that homeostatic properties of CD4+ cells reside in a sub-population of CD4+ CD45RBlo CD25+ T lymphocytes (Tregs) [76]–[78]. Transplanted CD4+ CD45RBlo CD25+ Tregs alone completely recapitulated the L. reuteri-induced, oxytocin-dependent phenomenon in Rag-deficient recipient mice. These novel associations with oxytocin may link the few previously published reports suggesting that upon birth oxytocin simultaneously up-regulates IFN-γ and CD25 expression culminating in robust yet tightly regulated immunity for establishing self vs. non-self [22]–[23], [50].

The association between Foxp3+ Tregs and oxytocin may involve a cellular trafficking role, based on lowered numbers of Foxp3+ cells in the wound sites of oxy-KO mice fed L. reuteri compared to wild type also fed L. reuteri. Notably, the increase in Foxp3+ Tregs in wounds after administration of L. reuteri in wild type mice and in wounds of mice receiving L. reuteri-primed Foxp3+ Tregs also indicate possible proliferative or stimulatory aspects for oxytocin. In Rag2-KO mice, still capable of producing oxytocin, adoptively transferred CD4+FoxP3+ Tregs from either oxy-KO or wild type donors fed L. reuteri will traffic to wound sites, but with performance deficits in oxy-KO cells. That exogenous oxytocin administration restores the ability of oxy-KO mice to heal their wounds supports the idea that trafficking and stimulation of Tregs in wound sites is potentially a direct consequence of circulating oxytocin levels. Extensive in vivo and in vitro characterizations of Treg biology under these various conditions is currently underway in our laboratory.

From an evolutionary perspective, we assert that lactic acid bacteria co-evolved with mammals partially by exploiting oxytocin to optimize reproductive fitness. As a regulator of immune competence, oxytocin may provide selective advantage to hosts in conditions where dedicating energy to immune function is vital: namely child-rearing and living in social or kin groups. Elevated plasma oxytocin levels in our separate studies correlated with improved maternal care and neonatal survival rates in mother C57BL/6 mice consuming L. reuteri (data not shown). Oxytocin has been inversely linked with post-partum depression and maternal neglect in human females [79], highlighting substantial maternal and infant gains during symbiotic microbial interactions. Benefits of this microbial synergy may extend beyond maternal-infant bonding, to social superiority with oxytocin enhancing cooperation within social groups while promoting aggression towards competitors [80]. These inter-related roles for oxytocin may impact a natural selection process favoring complex social organizations required for evolutionary success.

The combination of oxytocin with sex-linked physical, reproductive, and immune responses could have selective advantages in mammalian hosts – providing social functionality concomitant with improved immune function for survival in groups. In fact, in humans, specific SNPs in the 3′ untranslated region of the oxytocin receptor gene correlate with prosocial behavior, suggesting that modulation of these hormones at the levels of transcription and translation can have profound influence on these phenotypes [81]. Because microbes such as L. reuteri seem to modulate the expression of oxytocin, there is a clear role in co-evolution of microbial symbionts with their mammalian hosts for enhancing reproductive fitness and consequently also microbial proliferation.

Different impacts of L. reuteri on wound-healing according to gender may arise from the overlay of oxytocin with a sex-specific hormonal milieu. Female mice fed L. reuteri experience enhanced wound-healing over their male counterparts, suggesting that this microbe co-opts the endocrine environment of its host for effect. Interestingly, male wild type mice exhibited greater improvements in wound healing compared with male oxytocin-deficient mice, versus the female wild type mice compared to their oxytocin-deficient female counterparts. This observation suggests that females have alternative compensatory mechanisms in the absence of oxytocin in preparing the immune system for the wound healing process. We noted in our previous studies [16] that female mice showed a more robust response in terms of skin pH and fur luster than do male mice. This phenotype likely reflects the ability of sex-steroid hormones to influence immunological processes in a sexually dimorphic manner. Generally, females mount more robust humoral and cell-mediated immunity than males; in the absence of oxytocin, females may benefit from these inherent advantages in immunity over males in wound healing [82]. The loss of a key hormonal regulator of the immune response for males results in significant challenges during wound repair and likely in other cases requiring tight regulation of cell-mediated immunity.

Our findings also raise diverse benefits for orally administered microbes to lessen impairments during normal aging, imparting health resiliency typical of much younger individuals. Wound healing capacity directly translates to nearly every aspect of traditional health and medicine [20], [83]. We identify key mechanisms uniting social support networks with improved injury repair in healthful longevity [47]. Humans have cultivated and consumed similar food-grade organisms in fermented beverages and active yogurts for thousands of years, supporting a low-risk, high-impact population-based approach. This microbe-endocrine-immune linkage has the potential to reduce hospitalizations, improve healing, lower risk for certain cancers, and enhance healthful aging.

Experimental Procedures

Animals

C57BL/6 wild type (wt), oxytocin-deficient WT and knockout (KO) B6;129S-Oxttm1Wsy/J mice (purchased initially from Jackson labs; Bar Harbor, ME), C57BL/6 Foxp3EGFP which co-express eGFP and the regulatory T cell-specific transcription factor Foxp3 (Jackson labs), and 129SvEv Rag2-ko mice (Taconic Farms; Germantown, NY) were housed and handled in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities using techniques and diets including Lactobacillus reuteri as specifically approved by Massachusetts Institute of Technology's Committee on Animal Care (CAC) (MIT CAC protocol # 0912-090-15 and 0909-090-12). The MIT CAC (IACUC) specifically approved the studies as well as the housing and handling of these animals. Mice were bred in-house to achieve experimental groups. The B6;129S-Oxttm1Wsy/J mice were back-crossed eight generations to the 129 strain background prior to using for adoptive transfer experiments with Rag2-KO mice. Each experiment included 5-15 animals per group with one or two replications (total N = 10–30 mice examined per group) unless otherwise specified.

Mice underwent a standardized 2.0mm cutaneous biopsy procedure [76], while under general inhalant anesthesia, and with pre- and post-operative buprenorphine analgesia. Tissues for analyses were collected upon necropsy at specified time points. For the initial studies, C57BL/6 wt mice, receiving either L. reuteri treatment or regular water, underwent tissue biopsy at approximately six months of age, in order to mimic wound repair in middle-aged humans subjects, and were examined at three, six, or twelve days post-biopsy. For subsequent experiments, oxt-KO mice and their oxt-WT littermates entered experiments at 6-8 weeks of age. These mice underwent tissue biopsy at approximately 10–12 weeks of age, and were finally euthanized with CO2 and examined by morphometry as described below.

Special microbial treatments for animals

Mice were fed standard rodent chow (MRH 3000; Purina Labs, St Louis MO). Subsets of animals were supplemented orally with a strain of Lactobacillus reuteri [ATCC-PTA- 6475], originally isolated from human milk, and subsequently cultivated as described elsewhere [16], [34], using a supply dosage of 3.5×105 organisms/mouse/day continuously in drinking water. For the initial studies, C57BL/6 wt mice at 6 months of age received L. reuteri as above, for at least two weeks prior to skin biopsy. For subsequent studies, oxt-KO and their littermate oxt-WT mice began drinking L. reuteri organisms, as above, starting at 6–8 wks of age, and then underwent biopsy approximately 2–3 weeks later at 10–12 weeks of age. Drinking water was replaced twice weekly to minimize variability in microbial exposure levels. Sham control mice received an E. coli non-pathogenic lab strain of K12 at the same dosage of 3.5X105 organisms/mouse/day in drinking water, as specified. Other control animals received regular drinking water.

Measurement of plasma oxytocin levels

Whole blood was collected terminally by cardiac puncture under general anesthesia to obtain plasma. Blood was collected into pre-chilled 5 ml EDTA tubes with 250 KIU of apoprotinin, and refrigerated until processing. Plasma was isolated by centrifugation at 1800 g, 15 minutes, 4°C, and then stored in aliquots at −70°C. Plasma was then tested commercially (AniLytics,. Inc., Gaithersburg, MD). Euthanasia for mice was performed mid-day for all subjects (n = 10 per group) to minimize variability due to Circadian rhythms.

Vagotomy procedure

C57BL/6 wt mice (n = 8 per group) underwent vagotomy at 8 weeks of age. The vagotomy procedure used gaseous isoflourane anesthesia. Once a surgical plane of anesthesia is assured by toe pinch, the surgical site was prepared, and the stomach and lower esophagus were visualized by upper midline laparotomy. After visualization, both vagal trunks including all neural and connective tissue surrounding the esophagus and below the diaphragm was removed to transect all small vagal branches. Sham surgery did all procedures except to truncate the vagus nerve. All animals received pro-operative and post-operative buprenorphine analgesia. At least a two-wk recovery period was allowed prior to initiation of probiotics and skin biopsy surgery. [26]

Supplementation with exogenous oxytocin

Exogenous oxytocin was used to independently determine impact on wound healing. Mice (n = 6 per group) were injected intraperitoneally every 4–6 hours with 15 units/mouse of oxytocin (Bimeda-MTC Animal Health Inc., Cambridge, Canada) suspended in Lactated Ringers solution (LRS). Sham treated mice received LRS solution only on the same dosing schedule. Treatments began 24–48 hours prior to skin biopsy, and continued throughout until euthanasia three days later.

Skin biopsy procedure

Mice underwent a 2.0mm cutaneous skin punch biopsy procedure as described elsewhere[30], [35]while under general inhalant anesthesia, and with pre- and post-operative buprenorphine analgesia. Tissues for analyses were collected upon necropsy at specified time points of three days, six days or 12 days after biopsy.

Excision (biopsy) wound area and epithelial closure assessments

Formalin-fixed, routinely-processed, paraffinized, flat wounded skin tissues were used for wound area measurements before being embedded in paraffin blocks. Direct microscopy with a Nikon eclipse 50i microscope and a Nikon DS-5 M-L1 digital camera was used to examine and photograph wounds in paraffinized gross skin specimens. The wound areas were measured in images using the ImageJ image processing and analysis program (NIH, Bethesda, MD). Results were recorded as image pixels. In addition, histological sections showing the maximum wound epidermal gap were used to evaluate re-epithelialization. The epidermal gap was measured in low power x2 images. Results were recorded as pixels.

Assessment of wound healing rates

To calculate the wound healing rate constant, log-transformed wound area was fit using regression analyses. Slopes predicting the rate constants were compared with an interaction model with proc reg in SAS (alpha = 0.05) [84]–[85].

Adoptive transfer of T cells into recipient mice

CD4+ lymphocytes were isolated from C57BL/6 Foxp3EGFP which co-express eGFP and the regulatory T cell-specific transcription factor Foxp3 using magnetic beads (Dynal) for initial sorting, and then further sorted by flow cytometry to isolate gfp+ cells. In addition, CD4+ Treg lymphocytes isolated from 129 strain wild type or oxytocin-deficient mice used magnetic beads (Dynal) for initial sorting, and then were further sorted by hi-speed flow cytometry (MoFlow2) to obtain purified populations of CD4+ cells or CD4+CD45RBloCD25+ lymphocytes determined to be ∼96% pure as previously described elsewhere. In both models, Rag2−/− recipient mice were then injected intraperitoneally with 4X106 cells. All recipient animals were provided regular drinking water without supplementary L. reuteri and underwent skin biopsy at three weeks after cell transfer.

Histopathology and immunohistochemistry

Formalin-fixed wounded skin and mesenteric lymph node tissues were embedded in paraffin, cut at 4–5 µm, and stained with hematoxylin and eosin (HE). Step sectioning was applied in order to reach the proximity of the mid-wound level in skin samples. Then, multiple serial sections were taken from each sample for HE, Masson's trichrome and Toluidine blue stains and immunohistochemistry. Sections showing the maximum wound epidermal gap were used to evaluate re-epithelialization. The epidermal gap was measured in low power x2 images. Results were recorded as pixels. Immunohistochemistry (IHC) and morphometric assessment were as previously described[76]. IHC-positive immune cells and green-stained fibrous tissue with Masson's trichrome stain were counted in x20 images and results were recorded as number of cells or number of green collagen pixels per image. Primary antibodies for IHC included rabbit polyclonal antibodies for myeloperoxidase (ThermoFisher Scientific/Lab Vision, Fremont, CA), IL-17 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cleaved caspase-3 (Cell Signaling, Beverly, MA) and oxytocin-receptor (Abcam, Cambridge, UK), rabbit monoclonal antibodies for C-kit and Ki-67 (Cell Marque, Rocklin, CA) and a rat monoclonal antibody for Foxp3 (eBioscience, Inc., San Diego, CA) detection. Primary antibody binding was detected with goat anti-rabbit polymer HRP (ZytoChem Plus, Berlin, Germany) or biotinylated goat-anti rat IgG (Serotec, Oxford, UK). Heat-induced antigen retrieval was performed with citrate buffer, pH 6, for myeloperoxidase, caspase-3 and oxytocin-receptor, with EDTA buffer, pH 8, for C-kit and foxp3 detection or with CC1 epitope retrieval solution (Ventana Medical Systems, Inc., Tucson, AZ) for ki-67 and IL-17. The ImageJ image processing and analysis program (NIH, Bethesda, MD) was used for all quantitative histomorphometry assessments.

Statistical analyses

For all statistical analyses the Mann-Whitney U test (Graphpad Prism version 4.0 for windows, Graph-Pad software, San Diego, CA, USA) was used. Effects were considered to be significant at p<0.05. The y-axis of bar graphs stands for the mean±SEM of counts indicated in each graph.

Supporting Information

L. reuteri-induced accelerated wound repair exhibits key histopathological features at day 6. Control mice at day 6 post-wounding have incomplete re-epithelialization (arrow-head marks the leading edge of re-epithelialization). An area of the granulation tissue in the wound bed (inset) is shown bellow in higher magnification. The immature granulation tissue is loose, edematous and has emerging vessels and many activated (plump) fibroblasts (arrow), abundant neutrophils (black arrow-head ) and macrophages (white arrow-head). In contrast, the wounds of L. reuteri-treated mice at the same time-point show complete re-epithelialization and the granulation tissue in the wound bed is more mature. The boxed area is shown bellow in higher magnification. The mature granulation tissue has absent neutrophils and a chronic inflammatory component (lymphocytes-black arrow-head), elongated fibroblasts (black arrow-head) and early deposition of collagen fibers (white arrow-head). Hematoxylin and Eosin. Scale bars: upper panels low magnification = 250 µm; high magnifications of the inset areas (lower panels) = 25 µm.

Cellular proliferation in wounds at 6 days post-wounding. Proliferation features reflect the more advanced healing stage conferred by interconnected roles of (a) L. reuteri, (b) oxytocin and (c) regulatory T-cells. A large number of ki-67+ epidermal cells locate at the migrating edges of the epidermis in the open wounds of control experimental groups. The newly formed epidermis sealing the wounds of mice as a result of treatment has less proliferating cells. In the wound bed, early-stages of wound healing are characterized by the high proliferating activity of neutrophils (N) and fibroblasts (F). The more advanced stage of wound healing in animals receiving treatments is characterized by less proliferating fibroblasts. Immunohistochemistry: Diaminobenzidine chromogen, Hematoxylin counterstain. Scale bars = 50 µm.

Apoptosis in wounds at 6 days post-wounding. Similarly to proliferation, apoptosis in wounds (probed by Caspase-3-specific immunohistochemistry) also reflects the more advanced healing stage due to interrelated activity of (a) L. reuteri, (b) oxytocin and (c) regulatory T-cells. Although there are no caspase 3+ cells in the epidermis, increased numbers of apoptotic fibroblasts are seen in the granulation tissue bed only in the untreated experimental groups showing accelerated wound healing. Immunohistochemistry: Diaminobenzidine chromogen, Hematoxylin counterstain. Scale bars = 50 µm.